Alzheimer's disease (AD) afflicts 5 million Americans directly, and indirectly affects an estimated 13 million caregivers. Behind age, the second largest risk factor for AD is APOE (apolipoprotein-E) genotype where APOE4 carriers are at significantly higher risk for AD than their non-APOE4 (mainly APOE3) counterparts. We have discovered mimetics of apolipoprotein-E which are a family of small peptides derived from residues 133-149 of the holoprotein (apoE 133-149). These apoE-peptides display neurorestorative features that include anti-inflammatory and neuroprotective properties, and activities that stimulate the rebuilding of myelin (Li et al. 2006, Lynch et al. 2004, Hoane et al. 2007, Laskowitz et al. 2007, Tukhovskaya et al. 2008, Li et al. submitted, Christensen et al. submitted, Saransteva et al. revision submitted). In more complete mouse models of Alzheimer's disease that develop Alzheimer-like pathology, neuronal loss and behavioral deficits (Colton et al. 2006, Wilcock et al. 2008, Colton et al. unpublished), administration of these apoE-peptides reduced both amyloid plaque-like structures and neurofibrillary tangle- like structures; and significantly improved behavioral performance in a learning and memory task (please see Preliminary Results). We recently discovered that apoE-peptides function inside the cell to activate Protein Phosphatase 2A (PP2A) by a novel mechanism. This mechanism involves the binding of apoE-peptides to I2PP2A (Inhibitor #2 of PP2A) to form an apoE/I2PP2A complex that apparently is unable to inhibit PP2A phosphatase activity. Since Alzheimer's is a multi-component disorder involving loss of PP2A activity, an increase in I2PP2A, increased inflammation and increased levels of phospho-tau, we submit that apoE- peptide-mediated activation of PP2A to more healthy levels may have a significant and multi-dimensional advantage in slowing, halting and/or reversing the course of disease. The goal of this proposal is to find and develop orally active, small molecules that function like apoE- peptides to inhibit the I2PP2A protein, and thereby increase PP2A phosphatase activity. Thus, we propose to develop a high throughput screen based on competition of small molecules with apoE-peptide/I2PP2A complex formation. This competition assay will be used to screen a library of 100,000 diverse small molecules. Assuming a hit rate of 1%, we predict that e 100 small molecules will be identified in this competition assay that inhibit complex formation between apoE-peptide and I2PP2A. These 100 hits will then be assayed in a cell- based assay to assess their anti-inflammatory activity after treatment of cells with lipopolysaccharide (LPS) and each compound. Each compound that is confirmed to have anti-inflammatory activity will then be tested for its ability to activate PP2A phosphatase activity in whole cells The 5 most potent compounds will then be tested in whole animals for their ability to inhibit LPS stimulated cytokine release in a dose dependent fashion. Using our published method (Lynch et al. 2003), we will indirectly assay blood brain barrier penetration and brain activity of these 5 compounds. In future proposals, compounds that inhibit inflammation in the blood and in the brain, will be chemically modified with medicinal chemistry approaches and further evaluated for drug- like qualities in animal models of Alzheimer's disease. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) afflicts 5 million Americans directly, and indirectly affects an estimated 13 million caregivers. Behind age, APOE (apolipoprotein-E) genotype is the second largest risk factor: APOE4 carriers are at significantly higher risk for AD than their non-APOE4 (mainly APOE3) counterparts. We have discovered a family of small peptides that function as apoE mimetics. Derived from residues 133-149 of the apolipoprotein-E (apoE) holoprotein, these peptides display neurorestorative features that include anti- inflammatory and neuroprotective properties, and activities that stimulate the rebuilding of myelin (Li et al. 2006, Lynch et al. 2004, Hoane et al. 2007, Laskowitz et al. 2007, Tukhovskaya et al. 2008, Li et al. submitted, Chirstensen et al. submitted, Saransteva et al. revision submitted). These apoE mimetic peptides, in our pilot studies, reduced the presence of amyloid plaques, neurofibrillary tangles, and improved the learning and memory ability of our more complete transgenic mouse model of Alzheimer's disease. We now wish to find and develop small molecules that act in the same way as our apoE-mimetic peptides. This will require several levels of screening to find the few compounds that can be orally administered and show protective activity in the brains of experimental animals.